Balancing the functions of DNA extracellular traps in intracellular parasite infections: implications for host defense, disease pathology and therapy

The release of DNA to the extracellular milieu is a biological process referred to as etosis, which is involved in both physiological and pathological functions. Although the release of DNA extracellular traps (ETs) was initially attributed to innate immune cells such as neutrophils, eosinophils, and macrophages, recent studies have shown that T cells, as well as non-immune cells, are capable of releasing ETs. These structures were described primarily for their potential to trap and kill pathogens, presenting an important strategy of host defense. Intriguingly, these functions have been associated with intracellular pathogens such as the parasites Leishmania sp. and Trypanosoma cruzi, causative agents of leishmaniasis and Chagas disease, respectively. These are two devastating tropical diseases that lead to thousands of deaths every year. In an apparent contradiction, ETs can also induce and amplify inflammation, which may lead to worsening disease pathology. This has prompted the concept of targeting ETs’ release as a means of controlling tissue destruction to treat human diseases. What is the best approach to prevent disease severity: inducing ETs to kill pathogens or preventing their release? In this Perspective article, we will discuss the importance of understanding ETs released by different cell types and the need to balance their potentially complementary functions. In addition, we will explore other functions of ETs and their translational applications to benefit individuals infected with intracellular parasites and other pathogens. Ultimately, a better understanding of the role of ETs in disease pathogenesis will provide valuable insights into developing novel therapies for human diseases.


INTRODUCTION
Extracellular traps (ETs) are considered a form of cell death [1] and are mainly composed of DNA, proteins, and other cytoplasmic components released by cells. These structures have a diameter of 15-17 nm and globular domains of~25 nm. Transmission electron microscopy analysis of cross-sections of these traps revealed that they are not enclosed by membranes [2][3][4]. ETs are released by cells through a process known as "Etosis", which involves the activation of a series of intracellular signaling pathways leading to DNA de-condensation and its release into the extracellular environment [1,[5][6][7]. Considerable research has been conducted regarding ETs since their discovery, but we still do not fully understand the process of their formation and how to control their release in vivo.
DNA is the main component responsible for the ability of ETs to capture and trap microorganisms [2,38,39]. The proteins present in ETs include histones and enzymes [2,17,40] such as elastase, which can degrade the cell walls of captured pathogens, implicating ETs in their elimination [41]. Thus, trapping and killing pathogens, the very first function attributed to ETs, is a coordinated effort of their many components. The role of ETs in combating extracellular pathogens such as bacteria [2,15] and fungi [9,42] is clearly an important defense mechanism. But the release of these structures can also be triggered by intracellular pathogens such as viruses [43,44] and protozoan parasites [21,[45][46][47][48][49]. ETs can indeed trap and kill them while in their likely brief extracellular exposure. Despite significant advances in recent years in elucidating the release, composition, and functions of extracellular traps (ETs), the precise mechanisms underlying this process and the molecules that initiate their release remain incompletely understood. This knowledge gap is partly attributed to the diverse array of organisms that can activate ETs. These gaps represent critical areas of interest, as they offer potential avenues for developing novel strategies for controlling pathogenic infections and disease pathologies.
Comparing ET formation among infection models is challenging due to pathogen-specific and host-specific factors, limited data availability, and the lack of standardized methodologies. Standardization and collaborative research are crucial for advancing our understanding of ET formation in diverse infections. However, most studies on ETs have been conducted in neutrophils. These cells, in addition to being capable of forming extracellular DNA traps, are also capable of phagocytosing microorganisms. Therefore, the decision of neutrophils to generate NETs instead of phagocytosis is a crucial but still unknown point. This decision appears to be the result of a combination of multiple signals, including adhesive, metabolic, and activation conditions of the cells, environmental stimuli, and, importantly, the size and signals derived from the stimulating particle [50]. Some authors suggest that the size of the stimulating particle is important for the polarization of these two mechanisms [51]. It has been suggested that large particles, such as parasites, would induce NET formation, while small particles, such as bacteria and viruses, should be eliminated by phagocytosis. However, it has been demonstrated that both bacteria and viruses are capable of inducing cells to release ETs [2], while parasites, in addition to inducing NETs, can be phagocytized, as seen in studies with Leishmania sp and T. cruzi [21,[45][46][47][48][49]. The study by Sousa-Rocha D et al. in 2015 demonstrated that soluble Trypanosoma cruzi antigens as well as dead parasites are capable of inducing neutrophils to undergo Etosis [21]. Thus, although these parasites are obligatory intracellular pathogens, the interaction and activation necessary for NETosis occur mostly outside the cell. Table 1 summarizes the organisms in which the occurrence of ETs have been described, as well as the cellular source of ETs, and the stimulus that induced it formation.

ETS IN INTRACELLULAR PARASITE INFECTIONS
Most studies regarding the relationship of ETs and protozoan parasites were performed using Leishmania, the causative agent of leishmaniasis, a spectrum of diseases ranging from tegumentary to deadly visceral forms [52,53]. Leishmania is transmitted to humans through the bite of an infected hematophagous female phlebotomine sandfly during her blood meal [54]. Amongst the tegumentary forms, cutaneous leishmaniasis is the most common manifestation and is characterized by single (localized, CL) or multiple (disseminated, DL) skin sores [55], while mucosal leishmaniasis (ML) mainly affects nasopharyngeal tissues [55]. These forms are mainly associated with L. braziliensis and L. amazonensis species in endemic areas of the Americas, where it is highly prevalent [56]. Visceral leishmaniasis (VL), caused mainly by L. donovani and L. chagasi, is the most severe form of the disease and can be fatal if not diagnosed early, and properly treated. It is estimated that there are 30,000 new cases of VL and over 1 million new cases of CL each year [57], and that more than 1 billion people are at risk of infection [58]. These diseases disproportionately affect economically and socially vulnerable populations, causing significant societal and economic impacts. Therefore, concerted efforts toward their control are of utmost importance.
Regardless of the species of Leishmania, two main stages of the parasite have been defined: amastigotes and promastigotes. Amastigotes typically reside inside the macrophages of the vertebrate host, while promastigotes are found mainly in the phlebotomine vector [57], and are the form transmitted during the sandfly's bloodmeal.
The first report of the interaction between extracellular traps (ETs) and Leishmania sp. demonstrated that L. amazonensis promastigotes were ensnared in DNA, elastase, and histonecontaining neutrophil extracellular traps (NETs), which exhibited leishmanicidal properties [46]. Moreover, immunofluorescence analysis of biopsies from patients with CL infected with L. amazonensis indicated the presence of DNA and elastasecontaining structures, suggestive of NETs in vivo [46]. This finding was confirmed in a subsequent study by Morgado et al. [48]. Subsequent studies have revealed the crucial role of PI3Kinase isoforms in L. amazonensis-induced NETosis [59]. Specifically, it was demonstrated that PI3Kγ activates a reactive oxygen species (ROS)-dependent NETosis, whereas PI3Kδ induces a ROS-independent pathway regulated by intracellular calcium. These findings point to the potential of targeting the PI3K pathway as a strategy to control NET formation triggered by L. amazonensis.
It is interesting to note that while L. amazonensis is vulnerable to NETs, L. infantum is resistant to them. Although L. infantum is capable of inducing NET release, it can evade NET-mediated killing via 3′-nucleotidase/nuclease activity, revealing a new function for this enzyme [45]. It is unclear whether the susceptibility or resistance of L. amazonensis and L. infantum, respectively, to NET-mediated killing is directly linked to disease severity. Nevertheless, it is worth noting that the susceptible L. amazonensis is associated with milder forms of leishmaniasis, whereas the resistant L. infantum causes the severe and potentially fatal VL. Interestingly, molecules related to NETs are differentially regulated at different stages of L. infantum infection, with significant differences observed between patients with visceral leishmaniasis and asymptomatic individuals. These observations suggest that NETs may have distinct roles depending on the clinical stage of infection and may provide useful biomarkers for better characterizing asymptomatic infections in endemic regions [60].
The observation of ETs in lesions of CL and ML patients caused by L. braziliensis was a surprising finding, given the low number of polymorphonuclear cells and the predominance of mononuclear infiltrates in these lesions [61]. Koh et al. demonstrated the presence of CD8-derived ETs in lesions from patients with CL and ML. These ETs were found to co-localize with CD107+ vesicles and were correlated with disease progression and severity. In vitro studies showed that CD8-derived ETs contained CD107+ vesicles and, in a live video, were observed to mediate the death of neighboring cells. This study proposed a novel function for CD8-derived ETs, namely, the delivery of cytotoxic granules to target cells, suggesting a new mechanism of cytotoxicity that operates independently of cell-to-cell contact [25].
Recent studies demonstrated that the saliva of the Leishmania sp. vector, Lutzomyia longipalpis, contains a potent nuclease that digests NETs, thereby enabling parasites to escape NET-mediated killing [62]. Conversely, another study by Gabriel and colleagues showed that NETs may contribute to the retention of L. donovani promastigotes at the site of inoculation, facilitating their uptake by mononuclear phagocytes [63].
Trypanosoma cruzi, a protozoan that causes Chagas disease (CD), which affects millions of people worldwide, mainly in Latin America [57], is another intracellular parasite that can trigger the release of ETs. T. cruzi belongs to the kinetoplastid family, the same family as Leishmania. T. cruzi causes a lifelong infection, and at least 30% of infected individuals develop one of the most severe heart diseases reported, which leads to thousands of deaths and disabilities annually [64]. While blood transfusion, organ transplantation, infected food, and mother-to-child transmission are important forms of transmission, T. cruzi is mainly transmitted by contact with the contaminated excreta of a triatomine vector [65]. Trypomastigotes, the infective form, are internalized by several host cells, including monocytes and muscle cells, and transform into amastigote forms. These forms replicate and differentiate back into trypomastigotes, rupturing the cells and being released to be internalized by other cells [66].
T. cruzi, like Leishmania, can induce the release of NETs, which are composed of DNA, histones, and elastase [46]. This release of NETs was shown to be dose and time-dependent and also required the generation of reactive oxygen species. It was found that antibodies against Toll-like receptors 2 and 4 decreased the release of NETs, and both live and dead parasites were able to induce their release. Interestingly, the induction of NETs increased the number of amastigotes, suggesting that it may influence increasing parasite replication or decreasing the release of trypomastigote forms. These findings provide new insights into the interaction between parasites and NETs and suggest that contact with NETs during Chagas disease may limit infection by affecting the parasite's infectivity and pathogenicity [21].
T. cruzi also induces ET formation by dog and opossum neutrophils. While the NETs were decorated with the protease elastase, it was suggested that the parasite efficiently evades ETmediated killing since T. cruzi can survive in these hosts for years [31]. The saliva of blood-feeding arthropods, which include the triatomine vector of T. cruzi, contains proteins that exhibit highaffinity binding to prostanoids such as TXA2. In vitro studies have shown that these proteins can prevent platelet-mediated NET formation and may contribute to antithrombotic effects in vivo [67].
The pathology associated with Chagas disease and several forms of leishmaniasis is predominantly inflammatory. Koh et al. found a significant correlation between CD8-derived ETs and the progression and severity of tegumentary leishmaniasis. The frequency of CD8-derived ETs was higher in ulcerated CL lesions compared to early non-ulcerated ones, and in ML lesions compared to CL lesions. The ML form is characterized by an intense, uncontrolled inflammatory response, with high expression of TNF and IFN-gamma, and low expression of IL-10 receptor by inflammatory cells [68]. It is possible that CD8-derived ETs induced and exacerbated the inflammatory reaction and tissue destruction, but further research is needed to confirm this hypothesis. Analysis of the inflammatory infiltrate present in the myocardium of Chagas disease cardiomyopathy patients has shown an abundance of CD8+ cells expressing cytotoxic molecules and inflammatory cytokines [69][70][71]. However, it remains unclear if these CD8 cells or any other cell type in the infiltrate can release ETs. Importantly, previous research has shown a link between ETs and cardiovascular diseases such as atrial fibrillation [72], acute myocardial infarction [73], and hypertrophic remodeling of the myocardium [74], indicating that this mechanism could also be involved in Chagas disease.

TARGETING ETS TO TREAT HUMAN DISEASES
The formation and release of extracellular traps (ETs) are complex cellular processes that involve the activation of various intracellular signaling pathways often associated with the inflammatory response. For instance, the activation of phosphoinositide 3-kinase (PI3K) and the generation of reactive oxygen species (ROS) have been implicated in this process [75]. The DNA present in ETs can stimulate specific receptors present in immune cells, including Toll-like receptor 9 (TLR9), which can trigger a signaling cascade leading to the production of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α [76,77]. Histones, which are also present in ETs, can engage membrane receptors and activate immune cells, thus contributing to the inflammatory response and inducing the production of inflammatory cytokines [78]. Therefore, ETs are involved in the inflammatory immune response, and their excessive release can lead to chronic inflammation and tissue damage. For example, in cases of sepsis, a severe infection that can lead to multiple organ failure, excessive ETs release can contribute to the destruction of surrounding tissues [79]. Similarly, in parasitic diseases such as those discussed above, the release of ETs can lead to chronic inflammation and tissue damage. Hence, a thorough understanding of the mechanisms underlying ET formation and release is essential to identify potential therapeutic targets for the treatment of inflammatory diseases.
Several therapeutic approaches have been considered to modulate the effects of ETs. Table 2 summarizes some of the strategies that have been employed to inhibit the production or release of ETs, showing their mechanism of action and potential applications. However, it is important to note that while controlling the activation of ET-releasing cells through inhibition of inflammatory signals is a valid approach, these control strategies should ideally act locally to better target the ETs themselves and prevent their activities. Moreover, some studies have questioned whether the generation of ETs is a physiological event necessary for biological functions since they may also occur spontaneously in the absence of specific stimuli [22]. Therefore, it is crucial to evaluate the impact of ET inhibition on both physiological and pathological processes to avoid unintended consequences. Another important consideration is that the majority of inhibitors were evaluated to impede the formation of extracellular traps (ETs) specifically by neutrophils. Given that ETs can be released by various cell types, it is crucial to ascertain whether these inhibitors would exhibit an inhibitory effect on the release of ETs by other cell types.

CONCLUDING REMARKS
The parasites T. cruzi and Leishmania sp. have undergone coevolution with mammalian hosts for millions of years, acquiring sophisticated mechanisms to evade the host's immune responses and persist in host tissues for prolonged periods. As a result, these parasites can cause chronic and debilitating diseases that significantly impact human health. Unfortunately, no vaccines for these diseases exist, and the available therapies are often limited by parasite resistance and serious side effects [80][81][82][83]. ETs possess both the ability to eliminate pathogens and to induce inflammation and tissue destruction, as demonstrated in Fig. 1, through complex cell activation mechanisms and functions. This concurrent occurrence of apparently opposing functions-parasite control and tissue destruction-prompts the question of whether to induce or inhibit the release of ETs to control infections and their consequences. Early ET release may benefit the host by clearing the pathogen, but interventions to control inflammation and pathology must be introduced subsequently. It is essential to conduct further research to determine the best timing for intervention and address critical questions such as: how parasites use ETs to evade host defenses, which specific molecules induce ET release in different diseases, whether this process depends on ligand-receptor interactions, and what are the consequences of inhibiting ET formation and release, given their potential physiological functions. Intracellular parasites are an excellent model for exploring these simultaneous and essential functions in these infections. By investigating the dual functions of  Fig. 1 Illustration of the etosis process, in which cells release extracellular traps to capture and eliminate microorganisms. This process can be performed by several types of cells, including neutrophils and macrophages. Microorganisms that can activate this process include bacteria, viruses, fungi and protozoa. Extracellular traps are composed of DNA, histones, and various proteins depending on the cell type and stimulus. One of their functions is to capture and eliminate microorganisms such as Leishmania sp. and T. cruzi. However, extracellular traps can also cause inflammation and tissue damage by stimulating the local production of cytokines and other proinflammatory molecules. Understanding these processes may help identify targets for therapeutic intervention, offering new alternatives to treat human diseases.